THE JOURNAL OF GENE MEDICINE RESEARCH ARTICLE J Gene Med 2008; 10: 81–93. Published online 15 November 2007 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/jgm.1120
Intracellular siRNA and precursor miRNA trafficking using bioresponsive copolypeptides
Ulrik L. Rahbek1,2 Kenneth A. Howard1,2 * David Oupicky3 Devika S. Manickam3 Mingdong Dong2 Anne F. Nielsen1 Thomas B. Hansen1 Flemming Besenbacher2 Jørgen Kjems1 1
Department of Molecular Biology, University of Aarhus, DK-8000, Denmark 2 Interdisciplinary Nanoscience Center, University of Aarhus, DK-8000, Denmark 3
Department of Pharmaceutical Sciences, Wayne State University, Detroit, MI 48202, USA *Correspondence to: Kenneth A. Howard, Department of Molecular Biology, iNANO, University of Aarhus, Building 1130, C.F. Møllers All´e, DK-8000, Aarhus C, Denmark. E-mail:
[email protected]
Abstract Background Small interfering RNAs (siRNAs) can induce specific gene silencing through cytoplasmic mRNA cleavage and nuclear transcriptional silencing, necessitating delivery to different cellular compartments. This study presents a reducible copolypeptide (rCPP) carrier containing different molar ratios of a histidine-rich peptide (HRP) and nuclear localization sequence (NLS) peptide to modulate intracellular trafficking of transfected siRNA and primary RNA transcripts (pri-miRNA). Methods Polyplex formation using siRNA and rCPP was demonstrated using photon correlation spectroscopy and atomic force microscopy. Confocal and fluorescence microscopy were used to investigate cellular uptake and nuclear trafficking whilst endogenous enhanced green fluorescent protein (EGFP) knockdown in H1299 cells was evaluated using flow cytometry. Transcriptional gene silencing of endogenous EF1A was verified using real-time reverse-transcription polymerase chain reaction (RT-PCR) and primiRNA nuclear processing was demonstrated using Northern analysis. Results rCPP-based polyplexes showed rapid cellular uptake and low cytotoxicity. Labelled components revealed intact polyplexes after 2 h that exhibited directed movements consistent with endosomal trafficking. Polyplex-mediated knockdown of EGFP increased with greater HRP content. The inclusion of NLS promoted nuclear localization of transfected siRNAs and pri-miRNAs to the nuclear compartment allowing for transcriptional silencing of EF1A and Drosha and Dicer dependent expression of mature miRNA, respectively. Conclusion Our results demonstrate that reducible copolypeptides can be used as carriers for the non-toxic cellular delivery of siRNA and pri-miRNA. The nuclear targeting of rCPPs can be utilized for delivery of siRNAs and pri-miRNAs to the nuclear compartment for transcriptional gene silencing or endogenous processing into mature miRNA, respectively, which could potentially lead to improved therapeutic approaches. Copyright 2007 John Wiley & Sons, Ltd. Keywords
copolypeptides; polyplexes; siRNA; pri-miRNA; nuclear trafficking
Introduction Received: 25 May 2007 Revised: 31 August 2007 Accepted: 17 September 2007
Copyright 2007 John Wiley & Sons, Ltd.
Small interfering RNAs (siRNAs) are 21–25 nucleotide duplexes that have been found to induce silencing of specific genes through the mechanism of RNA interference (RNAi) [1,2]. In RNAi one strand of the duplex base pairs with a complementary mRNA strand and through the action of the
82
RNA-induced silencing complex (RISC) induces cleavage of the mRNA. This process is highly specific allowing targeting and silencing of single genes upon introduction of siRNAs into cells and has been used extensively as a tool for investigating cellular processes [3,4]. Originally thought to have been evolved as defence against viral infections, the discovery of a closely related class of endogenously expressed small RNAs, the microRNAs (miRNAs) of ∼22 nucleotides, has proven that the mechanism of RNAi has more widespread functions in the cells where it play key roles in diverse regulatory pathways such as developmental timing, cell proliferation and tumorigenesis [5,6]. The miRNAs mediate their effects on post-transcriptional gene silencing in a manner very similar to the siRNAs, through the use of the RISC complex. In mammalian cells miRNAs are transcribed as long primary RNA transcripts (pri-miRNAs) in the nucleus which are sequentially processed into mature miRNAs [7]. The endogenous sequential processing of miRNA has been shown to improve the potency of gene silencing compared to externally introduced synthetic siRNAs [8] and avoids activation of the interferon system, which has complicated the use of siRNA [9]. In addition as a tool to investigate cellular processes and target validation in drug discovery, siRNA and miRNA can be potentially used as therapeutic drugs to silence genes implicated in viral pathogenesis, inflammatory conditions and cancer [3]. In order to maximize the therapeutic efficacy, however, several drug delivery barriers dependent on the route of administration have to be overcome. Extracellular barriers, including the mononuclear phagocyte system [10] and the endothelial lining of blood vessels, have evolved to protect the human organism from pathogens and foreign material. In addition, intracellular factors, including compartmentalization and the availability of RNA to interact with intracellular targets, must be considered. Poor release from the endosomal compartments and subsequent degradation by the lysosomes restrict translocation into the cytoplasm where the siRNA and miRNA direct target mRNA degradation or translational repression [11,12]. Recent findings have shown that siRNAs are capable of inducing transcriptional gene silencing (TGS) through involvement of DNA methylation [13,14] and that siRNAs mediate knockdown of nucleus restricted transcripts [15–17]. In order to engage the nuclear RNAi pathways, the siRNA must enter the nucleus, a translocation process restricted by the nuclear envelope. Synthetic polycation-based vectors (polyplexes) such as polyethylenimine (PEI) or poly-L-lysine (PLL) have been widely used for in vitro and in vivo delivery of nucleic acids as a safer alternative to viral delivery systems [18–20]. The effectiveness of these polyplexes has been shown to increase with increased polymer molecular weight; however, increased cytotoxicity is encountered [21]. ‘Bioresponsive’ polycations containing reducible disulfide bridges that respond to intracellular redox conditions have proven advantageous in delivering nucleic acids into cells [22–25]. Upon cellular internalization, Copyright 2007 John Wiley & Sons, Ltd.
U. L. Rahbek et al.
the intracellular reducing environment facilitates polymer breakdown into low molecular weight components that exhibit reduced cytotoxicity. Furthermore, polymer breakdown activates polyplex decomplexation and consequent release of free nucleic acids allowing interaction with their intracellular targets [26]. Development of a non-toxic polyplex system capable of modulating RNA delivery and facilitating release within cytoplasmic and nuclear compartments will clearly improve the therapeutic potential of RNA-therapeutics. In this study we use a reducible copolypeptide (rCPP) composed of different molar ratios of a histidine-rich peptide (HRP) and a nuclear localization sequence (NLS) peptide. The HRP contains three lysine residues which due to their protonation at neutral and acidic pH are able to bind the negatively charged phosphate backbone of nucleic acids through electrostatic interactions. Six histidyl residues were included to promote endosomal escape due to the buffering capacity of histidyl residues in the endosomal and lysosomal pH range [27]. In order to allow targeting of the nuclear compartment a NLS peptide derived from the importin α binding SV40 large T antigen was included in various amounts [28]. This work describes formulation and physicochemical characterization of siRNA and miRNA polyplexes using the rCPP carrier containing different molar ratios of the HRP and NLS peptide and the ability of the complexes to modulate intracellular siRNA and pri-miRNA trafficking, transcriptional gene silencing and enhanced green fluorescent protein (EGFP) knockdown.
Materials and methods Materials Dulbecco’s modified Eagle’s medium (DMEM) and RPMI 1640 culture medium +GlutaMAX , DMEM without riboflavins and phenol red, penicillin/streptomycin (Pen/Strep), G418 selection factor, trypsin-EDTA (1×), foetal bovine serum (FBS) and 10 × TBE buffer were purchased from Invitrogen Corporation (Carlsbad, USA). CellTiter 96 AQueous One Solution cell proliferation assay (MTS) was purchased from Promega Corp. (Madison, USA). TransIT-TKO and TransITOligo transfection reagents were obtained from Mirus Corp. (Madison, WI, USA). An EGFP-specific siRNA duplex (Dharmacon, Boulder, CO, USA) containing the sequence: passenger, 5 -GACGUAAACGGCCACAAGUUC3 , guide, 3 -CGCUGCAUUUGCCGGUGUUCA-5 and an EGFP-mismatch siRNA duplex containing the sequence: passenger, 5 -GACGUUAGACUGACAAGUUC-3 , guide, 3 -CGCUGAAUCUGACCUGUGGUUCA-5 was used for nanoparticle characterization, cytotoxicity and EGFP knockdown studies. An EGFP-specific siRNA labelled with a Cy5 fluorophore on the 5 end of the guide strand was used for cellular uptake and trafficking studies. EF1A promoter specific siRNA were purchased from DNA Technology A/S (Aarhus, Denmark) and contained the J Gene Med 2008; 10: 81–93. DOI: 10.1002/jgm
83
siRNA and pri-miRNA Delivery by rCPPs
sequence: passenger, 5 -AAGGUGGCGCGGGGUAAACUG3 and guide, 3 -UUCCACCGCGCCCCAUUUGAC-5’.
Cell lines The human lung cancer cell line (H1299) stably expressing EGFP (2 h half-life), kindly provided by Dr. Anne Chauchereau (CNRS, Villejuif, France), was grown in RPMI 1640 + GlutaMAX supplemented with FBS, Pen/Strep and G418 selection factor. Human cervical cancer cells (HeLa) were grown in DMEM + GlutaMAX supplemented with FBS and Pen/Strep. All cell lines were incubated at 37 ◦ C in a 5% CO2 humidified environment.
Preparation of reducible polycations and polyplex formation The HRP, CKHHHKHHHKC, and the NLS peptide, CGAGPKKKRKVC, were synthesized and purified and reducible copolypeptides (rCPPs) prepared through oxidative copolymerization. Five rCPPs containing different amounts of NLS were prepared by adding HRP and NLS peptide at different molar ratios in the reaction mix (Table 1). The synthesis, composition and molecular weight of the polymers have previously been described [29]. rCPP-D was labelled by succinimidyl ester conjugated Alexa488 according to the Invitrogen protocol. RNA or siRNA was added to 20 mM sodium acetate buffer adjusted to pH 5.0 using acetic acid (0.2 M). rCPP was added to the nucleic acids at the desired N : P ratio and mixed by gently pipetting. Polyplexes were allowed to form for at least 1 h at room temperature prior to use. To calculate specific N : P ratios (defined as the molar ratio of reducible polycation amino groups/RNA phosphate groups) a mass per phosphate of 325 Da was used for RNA. The mass per charge of the rCPPs is listed in Table 1. The hydrodynamic diameter of the polyplexes was determined by photon correlation spectroscopy (PCS) using a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK). PCS was performed at 25 ◦ C in sodium acetate buffer in triplicate with sampling time and analysis set to automatic.
Stability of polyplexes under redox conditions using polyacrylamide gel electrophoresis
37 ◦ C were analyzed by electrophoresis using a 10% polyacrylamide gel (50 mM Tris-borate, pH 7.9, 1 mM EDTA) at 150–230 V for 2 h; they were then stained with ethidium bromide and visualized using a UV illuminator.
Determination of polyplex morphology using atomic force microscopy (AFM) Polyplex D (N : P ratio 10) with or without 25 mM DTT incubation for 30 min was deposited onto freshly cleaved micas for 10 min. After the solvent had evaporated, the sample was transferred for AFM. Freshly prepared samples were imaged using a commercial Nanoscope IV MultiMode atomic force microscope (Veeco Instruments, Santa Barbara, CA, USA) under ambient conditions. AFM imaging was performed in tapping mode at scan frequencies of 1–2 Hz with minimal loading forces applied and optimized feedback parameters. Several images were obtained from separate locations across the surfaces to ensure reproducibility. All the images were first flattened using the NanoScope software (Digital Instruments), while excluding the particles from the flattened area, and then analyzed automatically using the commercial Scanning Probe Image Processor (SPIP ) software (Image Metrology ApS, Lyngby, Denmark) to yield the particle diameter histograms from the images.
Cytotoxicity assay H1299 or HeLa cells were plated in a 96-well plate at a density of 4 × 104 cells/well and grown overnight. Medium was replaced by serum-free media and polyplexes and TransIT-TKO were added at a concentration of 50 nM siRNA/well for 4 h and then media was replaced by fresh growth medium. After incubation for 24 h, a CellTiter 96 AQueous One Solution cell proliferation assay (Promega, WI, USA) was used to investigate cytotoxicity. CellTiter proliferation assay solution was added to the wells and left for 3 h before absorbance was measured at 490 nm using a fluorescence plate reader.
Fluorescence microscopy
Polyplexes (N : P ratio 5 and 10) with or without prior incubation with 1,4-dithiothreitol (DTT) for 30 min at
HeLa or primary macrophage cells were grown in glass-bottomed dishes (MatTek Corp., MA, USA) or on glass coverslips in 12-well plates. In some experiments,
Table 1. Composition and weight per charge of the reducible copolypeptides Polymer Identity A B C D E
NLS peptide in reaction (mol%)
NLS peptide in polymer (mol%)
HRP peptide in reaction (mol%)
HRP peptide in polymer (mol%)
Weight per Charge
100 75 50 25 0
100 73 48 20 0
0 25 50 75 100
0 27 52 80 100
325 315 300 280 255
Copyright 2007 John Wiley & Sons, Ltd.
MW ∼100.000 ∼60.000 ∼200.000
J Gene Med 2008; 10: 81–93. DOI: 10.1002/jgm
84
HeLa cells were transiently transfected using TransITOligo to express cyan fluorescent protein (CFP)-TI-VAMP according to Mirus protocol. The pCFP-TI-VAMP plasmid was a generous gift from Dr Edouard Bertrand (IGMM, Montpellier, France). For live cell experiments transfected cells were incubated in phosphate-buffered saline (PBS) + 1 µg/ml Hoechst 33 342 (Molecular Probes) for 10 min, washed in PBS after which colourless DMEM + 5% FBS was added and the dish was sealed with Parafilm. Fluorescence images were obtained using a Zeiss Axiovert 200M microscope equipped with a heated stage and objective, a 100× 1.4 NA planapochromatic lens and a Coolsnap HQ camera (Ropers Scientific) and analyzed using Meta Morph software (Universal Imaging Corp.). For time lapse studies, a ‘No Neighbours’ deblurring algorithm was used to improve the signal-to-noise ratio (AutoQuant Deblur, Media Cybernetics). For fixed cell experiments, transfected cells were washed in PBS and fixed in 4% paraformaldehyde at room temperature for 10 min. Coverslips were then mounted onto glass slides using Prolong Gold mounting medium with 4 ,6diamidino-2-phenylindole (DAPI). Fluorescence images were obtained using a Zeiss LSM 510 Meta confocal laser scanning microscope equipped with a 100× 1.4 NA planapochromatic lens.
Gene silencing in an EGFP-expressing human cell line H1299 green cells were plated on 24-well plates (105 cells/well) 24 h prior to transfection. The medium was replaced with serum-free media and the polyplex formulations added at 50 nM siRNA per well. TransITTKO transfections were carried out according to Mirus protocol. After 4 h the media was replaced with fresh growth medium. The cells were left for 44 h and then resuspended in PBS containing 1% paraformaldehyde. The EGFP cell fluorescence was measured using a FACSCalibur flow cytometer (Becton Dickenson). A histogram plot with log green fluorescence intensity on the x-axis and cell number on the y-axis was used to define median fluorescence intensity of the main cell population defined by scatter properties (FSC, SSC, not shown).
Transcriptional gene silencing HeLa cells were plated in a 12-well plate 24 h prior to transfection. The medium was replaced with serumfree media and polyplexes were added at 50 nM EF1A promoter siRNA per well. TransIT-TKO transfections were carried out according to Mirus protocol. After 4 h the media was replaced with fresh growth medium. The cells were left for 48 h after which total RNA was isolated using TriZol Reagent (Invitrogen). RNA (1 µg) was used as template for the oligo-dT primed reverse transcription polymerase chain reaction (RTPCR) using a M-MLV RT kit (Invitrogen). The resultant Copyright 2007 John Wiley & Sons, Ltd.
U. L. Rahbek et al.
cDNA was used in a real-time PCR using the following primers: EF1A, F 5 -CTGAACCATCCAGGCCAAAT-3 , R 5 ATGTGAGCCGTGTGGCAA T-3 and GAPDH, F 5 GAAGGTGAAGGTCGGAGT, R 5 GAAGATGGTGATGGGATTTC. The real-time RT-PCR data were normalized to GAPDH controls from the respective samples.
Northern blotting HeLa cells were plated in a 12-well dish 24 h prior to transfection. The media was removed and replaced with serum-free media and TransIT-TKO or rCPP/PrimiRNA23A polyplexes added at 15 nM pri-miRNA23a per well. Following 4 h the media was replaced with 1 ml fresh media containing 10% FBS. After 44 h total RNA was isolated using TriZol Reagent (Invitrogen). Amounts of 3 µg were loaded in each lane on a 12% denaturing gel and the amount of resolved RNA in the lanes was checked by ethidium bromide staining. The RNA was then transferred to a Hybond-N+ blotting membrane (Bio-Rad) overnight and probed with an oligonucleotide complementary to miRNA 23a (5 -GGAAATCCCTGGCAATGTGAT-3 ). The blotting membrane was pre-hybridized for 2 × 1 h in Church buffer and hybridized overnight at 37 ◦ C after which it was washed in 1× SSC +0.1% sodium dodecyl sulfate (SDS) and exposed on a Molecular Imager FX (Bio-Rad) and analyzed using Quantity One software (Bio-Rad).
Results Physicochemical characterization The ability of the rCPPs to form polyplexes with siRNA (21-mers) and disassemble under redox conditions was investigated using five different types of polymers (A–E) containing a variable ratio of HRP and NLS peptides (Table 1). The molecular parameters including hydrodynamic size and surface charge, which can influence cellular interactions, were measured using dynamic light scattering. At an N : P ratio of 10 all rCPPs were able to form discrete particles below 270 nm (Table 2). The particles formed were monodisperse with polydispersity indices below 0.5 (data not shown). The ζ potential measurements showed that all polyplexes had a net positive charge greater than 20 mV, reflecting excess rCPP at this N : P ratio (Table 2). The size of polyplex D containing Cy5-labelled siRNA and Alexa488-labelled rCPP-D was in the same range as the unlabelled polyplex indicating that the fluorescent groups attached did not interfere with polyplex formation and subsequent trafficking experiments (see below). The ability of the polyplexes to decomplex in a reducing environment was examined using the electrophoretic migration of siRNA in a polyacrylamide gel shift assay (Figure 1) and AFM (Figure 2). The polyplexes were incubated with 25 mM DTT for 30 min at 37 ◦ C after J Gene Med 2008; 10: 81–93. DOI: 10.1002/jgm
85
siRNA and pri-miRNA Delivery by rCPPs
Table 2. Molecular parameters of rCPP-based siRNA polyplexes. D (Cy5) contains Cy5-sense labelled siRNA. D (A488/Cy5) contains Alexa488-labelled rCPP and Cy5-labelled siRNA. Results are presented as mean values ± SD (n = 3) Polyplex (rCPP/siRNA)
Hydrodynamic radius (nm)
ζ potential (mV)
A B C D E D (Cy5) D (A488/Cy5)
240 ± 13 231 ± 20 261 ± 15 213 ± 18 213 ± 34 200 ± 2 267 ± 13
22 ± 2 33 ± 1 32 ± 1 30 ± 1 21 ± 1 30 ± 1 27 ± 2
which they were loaded onto a gel and the amount of released siRNA assessed by ethidium bromide staining. From Figure 1 it can be seen that small amounts of siRNA were released (Figure 1A, wells 2–11) under non-reducing conditions suggesting that they are in a particulate form and retarded in the gel whereas reduction of the polyplexes by DTT caused a significant increase in the amount of siRNA released (Figure 1B, wells 2–11). The amount of released siRNA was quantified using signal intensity analysis on the gel bands (Figure 1C). Decomplexation was most ineffective for polyplexes containing the A-type polymers suggesting that the highly
basic nature of pure NLS polymers may increase complex stability. AFM analysis of the morphology of polyplex D revealed a monodispersed distribution of nanoparticles (Figures 2A and 2B). Reduction of the copolypeptide by DTT led to significant changes in morphology due to increased polyplex size (Figures 2C and 2D). From Gaussian fitting the average size was found to increase from 19.2 ± 6.4 nm to 32.4 ± 10.1 nm after DTT reduction which suggests decomplexation through a process of particle swelling, most likely due to electrostatic repulsive forces between hydrated polymeric chains during siRNA release. A population of very small complexes (
